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Mechanisms of Copying of Carcinogen-damaged DNA and RNA by Translesion Polymerases

跨损伤聚合酶复制致癌物损伤的 DNA 和 RNA 的机制

基本信息

批准号：
9886242
负责人：
F PETER Guengerich
金额：
$ 35.48万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9886242
关键词：
8-hydroxyguanosine Active Sites Address Adenine Base Pairing Binding Biochemical Biological Biological Assay Biological Process Bypass Cancer Etiology Carcinogens Catalysis Cell Extracts Cell Maintenance Cells Chemicals Collaborations Complex Crystallization DNA DNA Adducts DNA Damage DNA Modification Process DNA Primers DNA biosynthesis DNA-Directed DNA Polymerase Disease Enzymes Fibroblasts Genetic Genetic Materials Genetic Transcription Goals Hela Cells High Pressure Liquid Chromatography Homeostasis Human Human Activities Individual Investigation Ions Kinetics Laboratories Lead Lesion Link Maintenance Malignant Neoplasms Metals Molecular Mutation Nucleotide Excision Repair Nucleotides Oligonucleotides Physiological Polymerase Property Pyrimidine Dimers RNA RNA primers RNA-Directed DNA Polymerase Reaction Reporting Reverse Transcription Ribonucleases Ribonucleosides Ribonucleotides Roentgen Rays Series Site Specificity Structure System Testing Thymidine Tissues Titrations Tyrosine Work X-Ray Crystallography adduct base chemical carcinogenesis experimental study human DNA in vitro activity insight interest novel polymerization repaired screening sugar systemic autoimmunity tripolyphosphate

项目摘要

PROJECT SUMMARY Genetic integrity is important in the maintenance of cellular homeostasis. Chemical and physical damage is well-known with DNA but less extensively studied with RNA. Miscopying of damaged bases is known to contribute to mutations and to cancer and other diseases. A focus in this laboratory for more than two decades has been DNA polymerases and their interactions with damaged DNA. Recent studies in our lab and by others have shown that DNA polymerases sometimes incorporate ribonucleoside triphosphates (rNTPs). In this laboratory, at least two of the human (h) translesion DNA polymerases, hpol η and hpol κ, have been shown to have unexpected activities with both DNA and RNA templates, including reverse transcription, DNA priming, and transcription. These activities will be studied systematically, particularly with several common lesions known to be formed in DNA and hypothesized to also be present in RNA. It is hypothesized that specific steric and bonding features of these polymerases impart these novel properties and that these biological properties are operative in cells. Features of the proposed studies include: (i) Characterization of the activity of hpol η in transcription, reverse transcription, and DNA priming. Steady- state and pre-steady-state kinetic analysis will be done to identify catalytic specificity and rate-limiting reaction steps. A key feature of the work will be several adducts in DNA and RNA (7,8-dihydro-8-oxoG (8- oxoG), 1,N6-ethenoadenine, and (thymidine-thymidine) cyclobutane pyrimidine dimer (CPD)), including analysis of how hpol η inserts correct or incorrect bases opposite each of these in RNA templates. X-Ray crystallography will be used to define features of hpol η contributing to the observed catalytic properties. Another feature of the work is analysis of levels of individual RNA adducts. (ii) Some similar questions will be addressed with hpol κ, which has been demonstrated to have reverse transcription and DNA priming activities. hpol ι has been reported to be capable of inserting rNTPs opposite undamaged DNA, an abasic site, and 8-oxoG. The mechanism will be investigated using X-ray crystal structures of ternary complexes with native and adducted DNA templates. (iii) The biological relevance of the hpol η reactions with RNA templates and insertion of ribonucleotides into DNA will be tested by investigating ribonucleotide insertion by hpol η opposite CPD (considered the most natural substrate for hpol η) under physiological conditions including typical cellular concentrations of metals and ribo- and 2ʹ′-deoxyribo-nucleotides. The effect of ribonucleotides opposite CPD will be examined with RNase H2 and nucleotide excision repair systems, in that CPD levels have been related to the presence of ribonucleotides in DNA, which in term are linked to systemic autoimmunity. XPV fibroblast cell extracts and cells will be utilized, which differ from control cells in whether they contain hpol η. Collectively these studies will provide new insight into the mechanisms of how these important polymerases act and what their biological functions are.

项目总结